Project Summary/Abstract Enterobacteriaceae and Pseudomonas aeruginosa (subject of this grant application) sense damage inflicted to their cell wall by ?-lactam antibiotics. The sensing event is linked to cell-wall recycling, which generates cell- wall-based natural products known as muropeptides. Certain internalized muropeptides induce antibiotic resistance mechanisms. This process has led to obsolescence of many of the ?-lactam antibiotics against Gram-negative bacteria. These complex events are poorly understood, and are the subject of study in this grant proposal. Two Specific Aims are proposed. I propose to study the periplasmic complexes involving lytic transglycosylases (LTs), which turn over the cell wall for the purpose of recycling, in Specific Aim 1. We have documented that there are 11 LTs in P. aeruginosa, whose individual reactions on the cell wall are described in this grant application. These enzymes are proposed to be involved in complexes with other proteins within the periplasm, whose identities are not known and represent a major gap in our knowledge of cell-wall processes. Whereas all the functions of LTs are not understood, one would appear to be repair of cell wall upon exposure of bacteria to ?-lactam antibiotics. Bulgecin A, a natural product inhibitor of LTs, was shown to potentiate the activity of ?-lactam antibiotics. This takes place as bulgecin A impairs the function of the LTs. Specific Aim 2 proposes to study bulgecin A and its derivatives by a detailed microbiological and enzymological evaluation of the compound. I anticipate that the successful completion of this proposed science will not only lead to the elucidation of these important events regulating the cell wall, but also will identify opportunities for their interruption as a means to circumventing the elaborate mechanism of ?-lactam resistance that Gram-negative bacteria have evolved. The needs for novel strategies in treating Gram-negative bacterial infections have never been as accute within the past 50 years. The use of combinations of bulgecin A (or a variant thereof) and ?- lactam antibiotics holds a great promise, which deserves to be investigated.